Direct Delivery of Functional Proteins and Enzymes to the Cytosol

Jul 1, 2013 - We report here a general strategy for direct delivery of functional proteins to the cytosol using nanoparticle-stabilized capsules (NPSC...
0 downloads 4 Views 1MB Size
Download PDF
Rui Tang, Chang Soo Kim,† David J. Solfiell,† Subinoy Rana,† Rubul Mout, Elih M. Vela´zquez-Delgado, Apiwat Chompoosor, Youngdo Jeong, Bo Yan, Zheng-Jiang Zhu, Chaekyu Kim, Jeanne A. Hardy, and Vincent M. Rotello*

ARTICLE

Direct Delivery of Functional Proteins and Enzymes to the Cytosol Using Nanoparticle-Stabilized Nanocapsules Department of Chemistry, University of Massachusetts-Amherst, 710 North Pleasant Street, Amherst, Massachusetts 01003, United States. †C. S. Kim, D. J. Solfiell, and S. Rana contributed equally to this work. R.T., D.J.S., A.C., and V.M.R. conceived and designed the experiments. R.T., D.J.S., S.R., C.S.K., Z.J.Z., C.K., R.M., B.Y., E.M.V., and Y.J. performed the experiments. All authors analyzed and discussed the data. R.T. wrote the manuscript, with revisions by D.J.S., C.S.K., S.R., J.A.H., and V.M.R.

ABSTRACT Intracellular protein delivery is an important tool for

both therapeutic and fundamental applications. Effective protein delivery faces two major challenges: efficient cellular uptake and avoiding endosomal sequestration. We report here a general strategy for direct delivery of functional proteins to the cytosol using nanoparticle-stabilized capsules (NPSCs). These NPSCs are formed and stabilized through supramolecular interactions between the nanoparticle, the protein cargo, and the fatty acid capsule interior. The NPSCs are ∼130 nm in diameter and feature low toxicity and excellent stability in serum. The effectiveness of these NPSCs as therapeutic protein carriers was demonstrated through the delivery of fully functional caspase-3 to HeLa cells with concomitant apoptosis. Analogous delivery of green fluorescent protein (GFP) confirmed cytosolic delivery as well as intracellular targeting of the delivered protein, demonstrating the utility of the system for both therapeutic and imaging applications. KEYWORDS: nanoparticle . nanocapsule . intracellular protein delivery . supramolecular assembly . organelle targeting

P

rotein-based therapeutics are promising tools for numerous biomedical applications.1 Intracellular delivery of functional proteins to replace missing, dysfunctional, or poorly expressed proteins or antagonize key intracellular pathways is the fastest growing and promising arm in modern drug development. Protein-based biologics have provided new therapeutic avenues for cancer2,3 and have also been used to treat a range of disease states including inflammation,4 lysosomal storage diseases,5 and transient cerebrovascular disorders.6 In addition to therapeutics, direct cytosolic delivery of functional proteins provides a potential tool for important biological applications including imaging,7 signaling studies,8,9 and cellular10,11 and stem cell engineering.12 A major complication in the use of protein-based drugs is the difficulty of delivering the unmodified, functional protein in an TANG ET AL.

active conformation to the necessary site of action. Mechanical delivery methods, such as microinjection and electroporation, have been used in research for decades.13 These methods, however, are low-throughput and disruptive and require specialized equipment to mechanically/physically puncture membranes, limiting their utility for in vivo applications. Another promising approach for delivering proteins utilizes covalent carriers14,15 that require irreversible modifications of the protein cargo.16,17 However, these covalent modifications of protein may impact protein function by interfering with protein folding.18,19 Furthermore, covalent modification of proteins for delivery is not a general approach; in cases where this strategy works customized optimization protocols tailored to each specific protein are required.14 A major challenge in all the nonmechanical protein delivery strategies described above is access of the protein to the cytosol. VOL. XXX

’

NO. XX

’

* Address correspondence to [email protected]. Received for review February 26, 2013 and accepted July 1, 2013. Published online 10.1021/nn402753y C XXXX American Chemical Society

000–000

’

XXXX

A www.acsnano.org

ARTICLE Figure 1. Design and preparation of nanoparticle-stabilized capsules (NPSCs). (a) Schematic showing the preparation of the protein NPSC complex containing caspase-3 or GFP and proposed delivery mechanism. The oil was a 1:1 mixture of linoleic acid (LA) and decanoic acid (DA). (b) TEM image of the dried GFP-NPSC. (c) Dynamic light scattering (DLS) histogram of GFP-NPSCs indicating an average diameter of 130 ( 40 nm.

Most delivery strategies rely on endocytic mechanisms of cellular uptake.20 As a result, the delivered proteins are susceptible to degradation in the endosome/ lysosome and are also unable to access key subcellular structures and machineries required for most applications.15 Currently, prolonged incubation times, elevated concentrations of delivery agents, and lysosomotropic reagents (such as chloroquine) are required to increase the efficiency of endosomal escape of delivered proteins.21,22 Supramolecular carrier-based delivery methods are modular, are easy to formulate, and operate through reversible associations with target proteins.23 In noncovalent strategies, proteins and delivery vectors selfassemble, allowing the transport of unmodified proteins into the cell, and overcome the limitations of using covalent protein modification strategies.14,15 We have recently developed a nanoparticlestabilized capsule (NPSC) system for the delivery of hydrophobic drugs.24 These NPSCs rapidly released small-molecule payloads from their oil interior through a membrane fusion-like25,26 hydrophobic interaction with the cell membrane. We hypothesized that this vehicle could also be used for cytosolic protein delivery by incorporating functional proteins into the NPSC shell. In this report, we demonstrate rapid and efficient delivery of therapeutic and imaging proteins into the TANG ET AL.

cytosol of HeLa cells using NPSCs. Caspase-3 (CASP3) was chosen to demonstrate therapeutic delivery of an active, biomedically important enzyme. Delivery of CASP3 is a particularly stringent test of the efficacy of this approach, as caspases are delicate enzymes that would be susceptible to inactivation during the delivery process. CASP3 was efficiently delivered into cells, resulting in effective induction of apoptosis. Green fluorescent protein (GFP) was used to determine the intracellular distribution of delivered proteins. The delivered GFP was distributed throughout the cell with identical cellular distribution to that of endogenously expressed red fluorescent protein (RFP). Further proof of cytosolic access was demonstrated through efficient intracellular targeting of a GFP fusion protein to the peroxisome.27 RESULTS AND DISCUSSION Nanoparticle-Stabilized Capsule Fabrication. The protein NPSC (CASP3-NPSC and GFP-NPSC) complexes were generated using a convergent process (Figure 1a; see Methods), where the HKRK AuNPs (see Figure 1a for the structure) provided a dual-mode supramolecular stabilization of the capsule wall. Briefly, proteins and AuNPs were mixed and incubated at room temperature for 10 min. Template emulsions were formed by homogenizing AuNPs in phosphate buffer (5 mM, VOL. XXX

’

NO. XX

’

000–000

’

B

XXXX www.acsnano.org

TANG ET AL.

ARTICLE

pH 7.4) and oil. Protein NPSCs were formed by combining template emulsions and the protein AuNP mixture. Combined hydrogen-bonding and electrostatic interactions between the guanidinium moieties of the particles and the carboxylates of the oil28,29 pin the nanoparticles to the capsule surface. Lateral stabilization is provided through interactions of the cationic nanoparticles with the anionic proteins (GFP pI = 5.9, CASP3 pI = 6.1) to be transported (Figure 1a).30 The capsule size is well controlled and in a regime well suited for intracellular delivery,31 with GFP-NPSCs and CASP3-NPSCs possessing average diameters as determined by dynamic light scattering of 130 ( 40 nm and 140 ( 20 nm, respectively (Figure 1b and c, Figure S1). A fluorescence titration was performed to measure the binding constant (Ks) between HKRK AuNPs and GFP, which was found to be 9  107 M 1, indicating high affinity of HKRK AuNPs to the protein (Figure S2a). Screening of the electrostatic interactions through the use of 500 mM NaCl into the mixture of AuNPs with GFP at 1:1 molar ratio resulted in complete recovery of the GFP fluorescence (Figure S2b), indicating reversible electrostatic interactions between the HKRK AuNP and the protein. GFP is almost completely quenched upon complexation by AuNPs. NPSC formation, however, results in a partial restoration in fluorescence. This result indicates a change in the structure of the GFPAuNP complex when at the interface of the NPSCs. To further probe the nature of the interactions, the NPSCs were incubated in 0.5% Tween-20 and 500 mM NaCl solutions. The high-salt solution resulted in no change in fluorescence, while Tween-20 resulted in an increase in fluorescence (79%; Figure S3). These results suggest that other interactions are involved in NPSC assembly beyond simple electrostatics. For CASP3, enzyme activity was assessed after interaction with HKRK AuNPs. Activity assays in vitro showed that the interaction between CASP3 and HKRK AuNPs did not inhibit the enzymatic activity. In fact, HKRK AuNPs enhanced the CASP3 activity by 2.3 times, a phenomenon observed previously in other nanoparticle protein systems32 (Figure S4). While efforts were made to assess activity of CASP3 in the NPSCs, the biphasic nature of the system prevented effective measurement of catalytic efficiency. Therapeutic Protein Delivery with NPSCs. Effective use of therapeutic proteins for intracellular applications requires rapid delivery of the protein in the active form to the cytosol.33,34 CASP3 is a highly promising therapeutic protein candidate35 owing to its critical role in apoptosis.36 Most tumor cells do not undergo appropriate apoptosis, which leads to unhindered cell growth.37 In many tumor cells CASP3 function is blocked due to overexpression of inhibitor of apoptosis (IAP) proteins that directly inhibit caspase function, preventing apoptosis. Intracellular delivery of sufficient levels of active CASP3 into the cytosol of such tumor cells circumvents this blockage, allowing tumor cells to enter apoptosis. Delivery of active

Figure 2. Delivery of caspase-3 into HeLa cells. Cells were incubated for 1 h with (a) CASP3-NPSC, (b) NPSC without CASP3, and (c) only CASP3 without NPSC. Subsequently, cells were stained using Yopro-1 (green fluorescence) and 7-AAD (red fluorescence) for 30 min, and the overlapped images are presented as apoptotic. Individual channel images are shown in Figure S5. (d) Apoptosis ratios of the cells after CASP3 delivery. Scale bars: 100 μm. The error bars represent the standard deviations of three parallel measurements.

caspases is extremely challenging, however, due to the negative charge and heterotetrameric state of the protein, the susceptibility of the active site to oxidation and alkylation, and the fragile nature of the active site that is composed of four highly mobile loops. VOL. XXX

’

NO. XX

’

000–000

’

C

XXXX www.acsnano.org

ARTICLE Figure 3. Delivery of GFP into HeLa cells. (a) Confocal image showing GFP delivery into HeLa cells by NPSCs. (b) Confocal images showing the co-localization of delivered GFP with expressed mCherry in HeLa cell. (c) Flow cytometry results of HeLa cells treated with GFP-NPSCs (red) or GFP alone (blue) for 2 h, using untreated HeLa cells as the control (black). Scale bars: 20 μm.

Demonstration of effective delivery of active CASP3 was established through incubation of HeLa cells with CASP3-NPSCs. After 1 h incubation, 72.0 ( 5.5% of HeLa cells underwent apoptosis, confirmed by double staining with Yopro-1 (a dye to detect apoptotic cell nuclei)38 and 7-AAD (a dye used to detect membrane disruption)39 (Figure 2a). The NPSC itself (Figure 2b), CASP3 only (Figure 2c), or CASP3 with AuNPs (Figure S5d) induced minimal levels of cell apoptosis, demonstrating that CASP3-NPSCs deliver the protein in the active form required for therapeutic applications. GFP Delivery Using NPSCs. The effectiveness of the delivered caspase suggested access of the delivered protein to the cytosol. The capability of cytosolic delivery of NPSCs was verified using GFP. Imaging experiments were performed using live cells to provide an accurate determination of protein distribution inside the cell.40 After 1 h incubation of GFP-NPSCs with HeLa cells, followed by 1 h incubation in fresh media, GFP was observed evenly distributed throughout the cytosol and nucleus (Figure 3a). In comparison, no delivery was observed by mixing empty NPSCs and GFP, indicating that the NPSCs serve as a carrier for the delivery cargo and not solely as a “hole puncher” for membrane penetration (Figure S6). To further demonstrate the cytosolic distribution of GFP, NPSCs were used to deliver GFP into mCherry-expressing HeLa cells.41 Confocal images (Figure 3b and Figure S7) show GFP delivered via NPSCs to be evenly distributed TANG ET AL.

throughout the cytosol and nucleus (with the exception of nucleoli) of HeLa cells, with no perceptible difference in distribution with respect to mCherry. Co-administration of Hoechst 33342 (a family of blue fluorescent dyes used to stain nuclei) confirmed that the delivered GFP had gained access to the nucleus, but not to the interiors of nucleoli (Figure S6), consistent with the distribution of cellularly expressed mCherry. Dispersal of GFP in the nucleus indicated that GFP was successfully delivered not just to the cytosol but also to the nucleus by free diffusion through nuclear pores.42 Taken together, these results demonstrate that the GFP was successfully delivered into the cytosol in a freely diffusing fashion. Flow cytometry was used to quantify the efficiency of GFP delivery into cells using NPSCs. The results showed that GFP was delivered to 77 ( 5% of cells in the GFP-NPSCs group (Figure 3c), consistent with the apoptosis induction observed with the CASP3-NPSCs. As expected, no delivery was observed for either the cells treated with GFP alone or untreated. Only low levels of delivery (20.6 ( 3.8%) were observed for GFPHKRK AuNP complexes that were not in capsule form (Figure S9), with most of the delivered GFP remaining entrapped in late endosomes/lysosomes (Figure S10). The difference in GFP delivery efficiency and subcellular localization between the particle-only group and the NPSC group supports the hypothesis that the improved efficiency of the delivery process in the NPSC VOL. XXX

’

NO. XX

’

000–000

’

D

XXXX www.acsnano.org

ARTICLE

Figure 4. Live cell imaging of rapid GFP release into the cytosol of a HeLa cell by NPSCs. “0 min” label represents the starting point of release. The arrow indicates a GFP-NPSC at the cell membrane prior to delivery of payload. Scale bar: 20 μm.

group resulted from a membrane fusion-delivery process, in agreement with previous studies.24,43,44 The NPSCs showed little cytotoxicity at the concentration (29 pM) used for the delivery studies (Figure S11). Live cell video imaging was then performed to track the intracellular release of protein payloads by NPSCs (Figure 4 and Movies S1, S2). As shown in Figure 4 and Movie S1, GFP-NPSCs remain intact for the first few minutes after attaching to the cell, confirmed by the co-localization of GFP and the AuNPs. Then GFP was rapidly released into the cytosol within 4 min. As seen from Movie S2, GFP was rapidly delivered; however the GFP-NPSC was not taken up as an intact entity by the cell. Taken together, these results strongly support the membrane fusion mechanism of delivery while demonstrating that uptake does not occur through an endocytotic mechanism. Intracellular Targeting of Delivered Proteins. To further demonstrate the versatility of NPSC protein delivery for intracellular delivery and imaging, we fused a peroxisomal-targeting sequence 1 (PTS1) to GFP (Figure S12).45 HeLa cells with stable expression of RFP bearing a C-terminal peroxisomal-targeting sequence were used to provide a fluorescence label for the peroxisomes. After 1 h incubation with GFP-PTS1-NPSC complexes, followed by 1 h incubation with fresh media, the confocal images clearly confirmed complete colocalization of the GFP-PTS1 fusion protein with the RFP-labeled peroxisomes (Figure 5a and Figure S13). In contrast, GFP without the PTS1 motif was evenly distributed throughout the cell as previously described (Figure 5b). These results clearly demonstrate that the protein delivered by the NPSCs gained complete access to the cytosol and that proteins delivered by NPSCs are capable of targeting subcellular organelles

METHODS Protein NPSC Complex Preparation. The HKRK ligand protected AuNPs (HKRK AuNPs) were synthesized following a reported method.30 To make the protein NPSC complex, 2.5 μM HKRK AuNPs were incubated with 1 μM GFP in 30 μL of phosphate

TANG ET AL.

Figure 5. Peroxisome targeting in HeLa cells transfected with RFP-PTS1 plasmid. (a) Co-localization of GFP-PTS1 fusion protein with the peroxisomal indicator (RFP-PTS1) expressed by the cell. (b) No co-localization of GFP was observed without PTS1 motif. Scale bars: 20 μm.

with essentially identical localization behavior to cellularly expressed proteins.27 CONCLUSION We have demonstrated a rapid and efficient protein delivery strategy using nanoparticle-based supramolecular nanocapsules. This approach provides a generalized strategy for direct delivery of functional proteins in their native forms. The supramolecular structure of the NPSCs makes the system stable for delivery of proteins yet reversible for payload release. In contrast with particle-based protein delivery systems, this method is capable of overcoming the major challenge of endosomal sequestration and thus holds great promise for effective protein therapy and imaging applications. We demonstrated the efficiency of the system by delivering a functional therapeutic protein, CASP3, into target cells to induce apoptosis. Delivery of fluorescent proteins demonstrated cytosolic distribution identical to that of a cellularly expressed counterpart, with delivery versatility further demonstrated by subcellular targeting of proteins. Taken together, the ability to incorporate active proteins on the capsule shell and efficiently deliver them into the cytosol opens up new opportunities for protein replacement therapy, in vivo disease prognosis through imaging, cellular organelle labeling, and cellular engineering.

buffer (5 mM, pH = 7.4) for 10 min. Then, 1 μL of the mixture of linoleic acid and decanoic acid (molar ratio = 1:1) was mixed with 500 μL of phosphate buffer (5 mM, pH = 7.4) containing 1 μM HKRK AuNPs and agitated by an amalgamator at 5000 rpm for 100 s to form emulsions. Finally, the mixture of the protein

VOL. XXX

’

NO. XX

’

000–000

’

E

XXXX www.acsnano.org

Acknowledgment. This research was supported by the NIH (GM077173 and EB014277 to V.M.R. and GM80532 to J.A.H.). D.J.S. acknowledges an NIH CBI training grant (S11110000000104). E.M.V. acknowledges National Science Foundation training grants (S21000025700000 and DGE-0504485). The authors also appreciate D. Callaham for help with confocal imaging. Supporting Information Available: Additional experimental details and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES AND NOTES 1. Leader, B.; Baca, Q. J.; Golan, D. E. Protein Therapeutics: A Summary and Pharmacological Classification. Nat. Rev. Drug Discovery 2008, 7, 21–39. 2. Yarden, Y.; Sliwkowski, M. X. Untangling the Erbb Signalling Network. Nat. Rev. Mol. Cell Biol. 2001, 2, 127–137. 3. Foltopoulou, P. F.; Tsiftsoglou, A. S.; Bonovolias, I. D.; Ingendoh, A. T.; Papadopoulou, L. C. Intracellular Delivery of Full Length Recombinant Human Mitochondrial L-Sco2 Protein into the Mitochondria of Permanent Cell Lines and Sco2 Deficient Patient's Primary Cells. Biochim. Biophys. Acta-Mol. Basis Dis. 2010, 1802, 497–508. 4. Jo, D.; Liu, D. Y.; Yao, S.; Collins, R. D.; Hawiger, J. Intracellular Protein Therapy with Socs3 Inhibits Inflammation and Apoptosis. Nat. Med. 2005, 11, 892–898. 5. Beck, M. Therapy for Lysosomal Storage Disorders. IUBMB Life 2010, 62, 33–40. 6. Ogawa, T.; Ono, S.; Ichikawa, T.; Arimitsu, S.; Onoda, K.; Tokunaga, K.; Sugiu, K.; Tomizawa, K.; Matsui, H.; Date, I. Protein Transduction Method for Cerebrovascular Disorders. Acta Med. Okayama 2009, 63, 1–7. 7. Cai, Z.; Ye, Z.; Yang, X.; Chang, Y.; Wang, H.; Liu, Y.; Cao, A. Encapsulated Enhanced Green Fluorescence Protein in Silica Nanoparticle for Cellular Imaging. Nanoscale 2011, 3, 1974–1976. 8. Ma, Y.; Cai, S.; Lv, Q.; Jiang, Q.; Zhang, Q.; Sodmergen; Zhai, Z.; Zhang, C. Lamin B Receptor Plays a Role in Stimulating Nuclear Envelope Production and Targeting Membrane Vesicles to Chromatin during Nuclear Envelope Assembly through Direct Interaction with Importin B. J. Cell Sci. 2007, 120, 520–530. 9. Shah, D. A.; Kwon, S. J.; Bale, S. S.; Banerjee, A.; Dordick, J. S.; Kane, R. S. Regulation of Stem Cell Signaling by NanoparticleMediated Intracellular Protein Delivery. Biomaterials 2011, 32, 3210–3219.

TANG ET AL.

10. Gaj, T.; Guo, J.; Kato, Y.; Sirk, S. J.; Barbas, C. F., 3rd Targeted Gene Knockout by Direct Delivery of Zinc-Finger Nuclease Proteins. Nat. Methods 2012, 9, 805–807. 11. Patsch, C.; Kesseler, D.; Edenhofer, F. Genetic Engineering of Mammalian Cells by Direct Delivery of Flp Recombinase Protein. Methods 2011, 53, 386–393. 12. Zhou, H. Y.; Wu, S. L.; Joo, J. Y.; Zhu, S. Y.; Han, D. W.; Lin, T. X.; Trauger, S.; Bien, G.; Yao, S.; Zhu, Y.; et al. Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins. Cell Stem Cell 2009, 4, 581–581. 13. Zhang, Y.; Yu, L. C. Microinjection as a Tool of Mechanical Delivery. Curr. Opin. Biotechnol. 2008, 19, 506–510. 14. Lo, S. L.; Wang, S. Intracellular Protein Delivery Systems Formed by Noncovalent Bonding Interactions between Amphipathic Peptide Carriers and Protein Cargos. Macromol. Rapid Commun. 2010, 31, 1134–1141. 15. Gu, Z.; Biswas, A.; Zhao, M. X.; Tang, Y. Tailoring Nanocarriers for Intracellular Protein Delivery. Chem. Soc. Rev. 2011, 40, 3638–3655. 16. Foster, S.; Duvall, C. L.; Crownover, E. F.; Hoffman, A. S.; Stayton, P. S. Intracellular Delivery of a Protein Antigen with an Endosomal-Releasing Polymer Enhances Cd8 T-Cell Production and Prophylactic Vaccine Efficacy. Bioconjugate Chem. 2010, 21, 2205–2212. 17. Kaczmarczyk, S. J.; Sitaraman, K.; Young, H. A.; Hughes, S. H.; Chatterjee, D. K. Protein Delivery Using Engineered Virus-Like Particles. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 16998–17003. 18. Rudolph, G. B. R., Lilie, H.; Jaenicke, R. In Protein Function: A Practical Approach, 2nd ed.; Creighton, T. E., Ed.; Oxford: New York, 1997; p 64. 19. Bennion, B. J.; Daggett, V. The Molecular Basis for the Chemical Denaturation of Proteins by Urea. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5142–5147. 20. Melikov, K.; Chernomordik, L. Arginine-Rich Cell Penetrating Peptides: From Endosomal Uptake to Nuclear Delivery. Cell. Mol. Life Sci. 2005, 62, 2739–2749. 21. Wadia, J. S.; Stan, R. V.; Dowdy, S. F. Transducible TAT-HA Fusogenic Peptide Enhances Escape of Tat-Fusion Proteins after Lipid Raft Macropinocytosis. Nat. Med. 2004, 10, 310–315. 22. Cronican, J. J.; Thompson, D. B.; Beier, K. T.; McNaughton, B. R.; Cepko, C. L.; Liu, D. R. Potent Delivery of Functional Proteins into Mammalian Cells in Vitro and in Vivo Using a Supercharged Protein. ACS Chem. Biol. 2010, 5, 747–752. 23. Salmaso, S.; Caliceti, P. Self Assembling Nanocomposites for Protein Delivery: Supramolecular Interactions of Soluble Polymers with Protein Drugs. Int. J. Pharm. 2013, 440, 111–123. 24. Yang, X. C.; Samanta, B.; Agasti, S. S.; Jeong, Y.; Zhu, Z. J.; Rana, S.; Miranda, O. R.; Rotello, V. M. Drug Delivery Using Nanoparticle-Stabilized Nanocapsules. Angew. Chem., Int. Ed. 2011, 50, 477–481. 25. Doxsey, S. J.; Sambrook, J.; Helenius, A.; White, J. An Efficient Method for Introducing Macromolecules into Living Cells. J. Cell Biol. 1985, 101, 19–27. 26. Almofti, M. R.; Harashima, H.; Shinohara, Y.; Almofti, A.; Baba, Y.; Kiwada, H. Cationic Liposome-Mediated Gene Delivery: Biophysical Study and Mechanism of Internalization. Arch. Biochem. Biophys. 2003, 410, 246–253. 27. Terlecky, S. R.; Koepke, J. I. Drug Delivery to Peroxisomes: Employing Unique Trafficking Mechanisms to Target Protein Therapeutics. Adv. Drug Delivery Rev. 2007, 59, 739–747. 28. Wiskur, S. L.; Lavigne, J. L.; Metzger, A.; Tobey, S. L.; Lynch, V.; Anslyn, E. V. Thermodynamic Analysis of Receptors Based on Guanidinium/Boronic Acid Groups for the Complexation of Carboxylates, Alpha-Hydroxycarboxylates, and Diols: Driving Force for Binding and Cooperativity. Chem.;Eur. J. 2004, 10, 3792–3804. 29. Zafar, A.; Melendez, R.; Geib, S. J.; Hamilton, A. D. Hydrogen Bond Controlled Aggregation of Guanidinium-Carboxylate Derivatives in the Solid State. Tetrahedron 2002, 58, 683–690. 30. Ghosh, P.; Yang, X.; Arvizo, R.; Zhu, Z. J.; Agasti, S. S.; Mo, Z.; Rotello, V. M. Intracellular Delivery of a MembraneImpermeable Enzyme in Active Form Using Functionalized

VOL. XXX

’

NO. XX

’

000–000

’

ARTICLE

and HKRK AuNPs was diluted to 45 μL with phosphate buffer (5 mM, pH = 7.4) followed by the addition of 5 μL of the emulsion. The protein NPSC complexes were ready to use after 10 min incubation at room temperature. The concentration of NPSC was 0.29 nM, calculated according to a reported method.24 The final concentrations of HKRK AuNPs and GFP were 1.5 μM and 600 nM, respectively. GFP Delivery. A total of 60 000 or 240 000 HeLa cells or transfected HeLa cells were cultured in a 24-well plate or confocal dish, respectively, for 24 h prior to delivery. The cells were washed by cold phosphate buffer saline three times right before delivery. After the preparation of cells, GFP-NPSC complex solution (50 or 150 μL of the GFP-NPSC complex diluted by 450 μL or 1.35 mL of the DMEM without FBS, respectively) was incubated with the cells for 1 h in a 24-well plate or confocal dish, followed by incubation with fresh DMEM with 10% FBS for 1 h unless otherwise mentioned. To study the co-localization with Hoescht 33342 or Lysotracker, the reagent was introduced 20 min before the observation. CASP3 Delivery. The procedure of CASP3 delivery was similar to that of GFP delivery, except that 30 000 HeLa cells were incubated in a 24-well plate for 24 h prior to delivery. After the protein delivery, the cells were stained by Yopro-1 and 7-AAD for 30 min, followed by observation under a fluorescence microscope. The apoptotic ratio of each sample was calculated as the ratio of fluorescently stained cells over total 100 cells. Conflict of Interest: The authors declare no competing financial interest.

F

XXXX www.acsnano.org

32.

33.

34.

35.

36.

37. 38.

39. 40.

41.

42. 43.

44.

45.

ARTICLE

31.

Gold Nanoparticles. J. Am. Chem. Soc. 2010, 132, 2642– 2645. Maruyama, K. Intracellular Targeting Delivery of Liposomal Drugs to Solid Tumors Based on EPR Effects. Adv. Drug Delivery Rev. 2011, 63, 161–169. Jeong, Y.; Duncan, B.; Park, M. H.; Kim, C.; Rotello, V. M. Reusable Biocatalytic Crosslinked Microparticles SelfAssembled from Enzyme-Nanoparticle Complexes. Chem. Commun. 2011, 47, 12077–12079. Yoshikawa, T.; Sugita, T.; Mukai, Y.; Abe, Y.; Nakagawa, S.; Kamada, H.; Tsunoda, S. I.; Tsutsumi, Y. The Augmentation of Intracellular Delivery of Peptide Therapeutics by Artificial Protein Transduction Domains. Biomaterials 2009, 30, 3318–3323. Zelphati, O.; Wang, Y.; Kitada, S.; Reed, J. C.; Felgner, P. L.; Corbeil, J. Intracellular Delivery of Proteins with a New Lipid-Mediated Delivery System. J. Biol. Chem. 2001, 276, 35103–35110. Cho, K. C.; Jeong, J. H.; Chung, H. J.; Joe, C. O.; Kim, S. W.; Park, T. G. Folate Receptor-Mediated Intracellular Delivery of Recombinant Caspase-3 for Inducing Apoptosis. J. Controlled Release 2005, 108, 121–131. Zassler, B.; Blasig, I. E.; Humpel, C. Protein Delivery of Caspase-3 Induces Cell Death in Malignant C6 Glioma, Primary Astrocytes and Immortalized and Primary Brain Capillary Endothelial Cells. J. Neuro-Oncol. 2005, 71, 127– 134. Lowe, S. W.; Lin, A. W. Apoptosis in Cancer. Carcinogenesis 2000, 21, 485–495. Idziorek, T.; Estaquier, J.; De Bels, F.; Ameisen, J. C. Yopro-1 Permits Cytofluorometric Analysis of Programmed Cell Death (Apoptosis) without Interfering with Cell Viability. J. Immunol. Methods 1995, 185, 249–258. Zimmermann, M.; Meyer, N. Annexin V/7-Aad Staining in Keratinocytes. Methods Mol. Biol. 2011, 740, 57–63. Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.; Verbeure, B.; Gait, M. J.; Chernomordik, L. V.; Lebleu, B. Cell-Penetrating Peptides - a Reevaluation of the Mechanism of Cellular Uptake. J. Biol. Chem. 2003, 278, 585–590. Carroll, P.; Schreuder, L. J.; Muwanguzi-Karugaba, J.; Wiles, S.; Robertson, B. D.; Ripoll, J.; Ward, T. H.; Bancroft, G. J.; Schaible, U. E.; Parish, T. Sensitive Detection of Gene Expression in Mycobacteria under Replicating and NonReplicating Conditions Using Optimized Far-Red Reporters. PLoS One 2010, 5, e9823. Dingwall, C.; Laskey, R. A. Protein Import into the Cell Nucleus. Annu. Rev. Cell Biol. 1986, 2, 367–390. Henriques, S. T.; Costa, H.; Castanho, M. Translocation of Beta-Galactosidase Mediated by the Cell-Penetrating Peptide Pep-1 into Lipid Vesicles and Human Hela Cells Is Driven by Membrane Electrostatic Potential. Biochemistry 2005, 44, 10189–10198. Raagel, H.; Saalik, P.; Pooga, M. Peptide-Mediated Protein Delivery-Which Pathways Are Penetrable? Biochim. Biophys. Acta-Biomembr. 2010, 1798, 2240–2248. Gould, S. J.; Keller, G. A.; Hosken, N.; Wilkinson, J.; Subramani, S. A Conserved Tripeptide Sorts Proteins to Peroxisomes. J. Cell Biol. 1989, 108, 1657–1664.

TANG ET AL.

VOL. XXX

’

NO. XX

’

000–000

’

G

XXXX www.acsnano.org